The overall goal of this proposal is to establish the mechanism by which Insulin Receptor Substrate-2 (IRS2) promotes breast cancer metastasis. The novel hypothesis to be examined is that IRS2 supports tumor progression through its regulation of breast cancer stem cell (CSC) function. CSCs represent a sub-population of tumor cells that have the ability to self-renew and generate tumor heterogeneity. These cells are sufficient to initiate primary and recurrent tumor growth, as well as secondary metastatic tumor growth. Understanding the biology of breast CSCs is essential for identifying novel approaches to target this aggressive tumor cell population to effectively eradicate both chemoresistant and metastatic tumors. The applicant?s preliminary data establish IRS2 as a novel CSC gene that enhances self-renewal and they identify a domain within IRS2 that is essential for this regulation. The ability of IRS2 to promote self-renewal also depends upon its recruitment and activation of PI3K. The IRS proteins are essential downstream effectors of the insulin (IR) and insulin like growth factor-1 (IGF-1R) receptors. Both of these receptors have been implicated in cancer and although their involvement in regulating CSC function has been investigated in some cancer contexts, a rigorous analysis of the mechanisms by which these receptors regulate CSC function has not been addressed. Given that targeting either the IGF-1R or IR directly can cause significant metabolic disruption, there is a need to develop novel approaches to inhibit the activity of these receptors without disrupting their normal metabolic regulation. The applicant proposes that selectively disrupting functions of IRS2 that promote cancer progression without interfering with its role in normal metabolic homeostasis would be such an approach. The results obtained from the experiments outlined in this proposal will lay the groundwork for developing targeted approaches that could be used in combination with current therapies to target CSCs to treat resistant and metastatic breast tumors. To investigate the hypothesis that IRS2-dependent signaling in response to IR/IGF-1R activation enhances CSC self-renewal and promotes metastasis the applicant will: 1) Establish that IRS2 but not IRS1 contributes to the function of breast CSCs. The hypothesis that IRS2 enhances breast CSC function in vivo and that this regulation is essential for breast cancer metastasis will be examined; 2) Elucidate the mechanism by which IRS2 regulates CSC function. The hypothesis that IRS2 enhances CSC self-renewal by the recruitment of factor(s) to a unique region within the IRS2 C-terminal tail (SR) that cooperate with PI3K/AKT signaling to regulate CSC self-renewal and promote breast cancer metastasis will be examined; 3) Investigate the contribution of IR/IGF-1R signaling to IRS2-dependent regulation of CSCs. The hypothesis that the IR-A isoform preferentially initiates IRS2 signaling to promote stemness and this function is enhanced by hyperinsulinemia to promote tumor progression will be examined.